Fermentation Methods for Producing Steviol Glycosides with Multi-Phase Feeding

ABSTRACT

Disclosed are methods for producing steviol glycosides, such as rebaudioside D and rebaudioside M, using engineered yeast. The methods include at least two phases: first and second phases where a glucose-containing feed composition is provided to the medium in different modes of feeding in each phase, such as variable feeding and then constant feeding. The two phase feeding can result in a growth rate that is slower in the second phase than in the first phase, and consequently increased steviol glycoside production rates, reduced fermentation times, and reduced biomass concentrations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 62/168,372 filed May 29, 2015, herein incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted concurrently herewith as an ASCII text file entitled “CAR0212WO_Sequence_Listing.txt,” created on May 27, 2016, and having a size of 92 kilobytes. The sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD

The present invention relates to fermentation methods for producing steviol glycosides, fermentation compositions, and steviol glycoside composition produced by fermentation.

BACKGROUND

Sugars, such as sucrose, fructose and glucose, are utilized to provide a pleasant taste to beverages, foods, pharmaceuticals, and oral hygienic/cosmetic products. Sucrose, in particular, imparts a taste preferred by consumers. Although sucrose provides superior sweetness characteristics, it is caloric. Non-caloric or lower caloric sweeteners have been introduced to satisfy consumer demand, and there is desire for these types of sweeteners that have favorable taste characteristics.

Stevia is a genus of about 240 species of herbs and shrubs in the sunflower family (Asteraceae), native to subtropical and tropical regions from western North America to South America. The species Stevia rebaudiana, commonly known as sweetleaf, sweet leaf, sugarleaf, or simply stevia, is widely grown for its sweet leaves. Stevia-based sweeteners may be obtained by extracting one or more sweet compounds from the leaves. Many of these compounds are steviol glycosides, which are glycosides of steviol, a diterpene compound. These diterpene glycosides are about 150 to 450 times sweeter than sugar. Steviol glycosides differ from each other by sweetness power as well as other sensory features contributing to taste quality such as bitterness, lingering aftertaste and the like. See Kinghorn, A. D., Stevia: The genus Stevia, Taylor & Francis, London (2002).

Examples of steviol glycosides are described in WO 2013/096420 (see, e.g., listing in FIG. 1); and in Ohta et. al., “Characterization of Novel Steviol Glycosides from Leaves of Stevia rebaudiana Morita,” J. Appl. Glycosi., 57, 199-209 (2010) (See, e.g., Table 4 at p. 204). Structurally, the diterpene glycosides are characterized by a single core structure, steviol, and differ by the presence of carbohydrate residues at positions C13 and C19, as presented in FIGS. 2a-2k . See also PCT Patent Publication WO 20013/096420.

Typically, on a dry weight basis, the four major steviol glycosides found in the leaves of Stevia are dulcoside A (0.3%), rebaudioside C (0.6-1.0%), rebaudioside A (3.8%) and stevioside (9.1%). Other glycosides identified in Stevia extract include one or more of rebaudioside B, D, E, F, G, H, I, J, K, L, M, N, O, steviolbioside and rubusoside.

While the major steviol glycoside Reb A is commonly used as sweetener in beverage applications it has off-taste issues. More recently, there has been focus on certain minor steviol glycosides which have better taste properties. For example, rebaudioside M has higher sweetness intensity and is more potent than other steviol glycosides (e.g., see Prakash, I., et al. (2013) Nat. Prod. Commun., 8: 1523-1526, and WO 2013/096420). Rebaudioside D tastes about 200-220 times sweeter than sucrose and in a sensory evaluation it had a slow onset of sweetness and was very clean, namely sweeter overall than sucrose, less sweet lingering aftertaste compared to sucrose (e.g., see Prakash, I., et al. (2012) Int. J. Mol. Sci., 13:15126-15136).

Molecular techniques have been used to prepare recombinant organisms capable of synthesizing steviol glycosides via fermentation. For example, recombinant strains of Saccharomyces cerevisiae having multiple transgenes encoding enzymes involved in steviol glycoside synthesis have been used for the production of rebaudioside M and rebaudioside D (see, for example, WO2014/122227). However, current fermentation methods using recombinant organisms do not adequately provide desirable steviol glycoside production rates, and also are associated with generation of large amounts of biomass and longer fermentation times to achieve desired steviol glycoside titers.

SUMMARY

The present invention generally relates to methods for producing steviol glycosides using engineered yeast, as well as fermentation compositions, and fermentation products that include one or more steviol glycosides. Fermentation conditions of the disclosure can promote one or more of the following: increased steviol glycoside titers from the engineered yeast, increased cell activity including increased steviol glycoside production rates, increased yield, reduced fermentation times, and reduced biomass concentrations. In exemplary embodiments the methods can be used for the production of steviol glycosides such as rebaudioside M, rebaudioside D, rebaudioside A, rebaudioside B, and combinations thereof.

One embodiment of the invention provides a method for producing steviol glycoside(s), which includes:

-   -   (a) growing engineered yeast capable of producing one or more         steviol glycoside(s) in a medium, wherein the engineered yeast         is grown at one or more growth rate(s) (dilution rate(s)) within         a first range; and wherein a composition comprising glucose is         added to the medium according to a first mode; and     -   (b) fermenting the medium with the engineered yeast to produce         the one or more steviol glycoside(s), wherein during fermenting,         a composition comprising glucose is added to the medium         according to a second mode that is different than the first         mode, and during fermenting the yeast grow at one or more growth         rate(s) (dilution rate(s)) within a second range, wherein the         second range is less than the first range.

Another embodiment of the invention provides a method for producing steviol glycoside(s), which includes:

at least steps (a) and (b) that involve the growth and fermentation of engineered yeast. In step (a) (i.e., a first phase) engineered yeast capable of producing one or more steviol glycoside(s) are grown in a medium at one or more growth rate(s) (dilution rate(s)) within a first range. Also in step (a) a composition comprising glucose is added to the medium according to a first mode that causes the yeast to grow within the first range. In step (b) (i.e., a second phase) the engineered yeast are fermented to produce the one or more steviol glycoside(s) where a composition comprising glucose is added to the medium according to a second mode that is different than the first mode. During step b), adding according to the second mode causes the yeast grow at one or more growth rate(s) (dilution rate(s)) within a second range which is less than the first range.

In an exemplary method, the yeast have a growth rate in step (a) in the range of about 0.06 h⁻¹ to about 0.15 h⁻¹, and a growth rate in step (b) in the range of about 0.015 h⁻¹ to about 0.09 h⁻¹. The change in growth rate from step (a) to step (b) can be caused by a change in “mode” of addition, such as by changing the rate of addition of a glucose-containing composition to the media, or changing how the glucose-containing composition is added to the media, such as providing a non-constant rate of feeding in step (a) and then a constant rate of feeding in step (b).

In another exemplary method, the engineered yeast are grown to a biomass amount in the range of 5 g dcw/L to 60 g dcw/L in step (a) and then to a biomass amount that does not exceed 150 g dcw/L in step (b).

In still other exemplary methods, the engineered yeast are grown by controlling the glucose feed rates based on a Respiratory Quotient (RQ), oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) or combinations thereof. In some exemplary methods, the glucose is adjusted during the fermentation phase to an RQ that is within a range of from about 0.5 to about 2.0.

The invention also provides a fermentation medium comprising steviol glycoside(s) obtained according to the method of the disclosure, and also a steviol glycoside composition obtained from the fermentation medium.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representative mevalonate pathway.

FIG. 2 shows a representative non-mevalonate pathway.

FIG. 3 shows a representative pathway for steviol production.

FIG. 4 shows representative pathways for the biosynthesis of steviol glycosides from steviol.

DETAILED DESCRIPTION

Embodiments of the disclosure described herein are not intended to be exhaustive or to limit the invention to the precise forms disclosed in the following detailed description. Rather a purpose of the embodiments chosen and described is so that the appreciation and understanding by others skilled in the art of the principles and practices of the present invention can be facilitated.

Fermentation methods of the disclosure use engineered yeast capable of producing steviol glycosides. An engineered yeast capable of producing steviol glycosides can include one or more exogenous nucleic acids that encode enzyme(s) that promote formation of one or more steviol glycosides in the cell.

As used herein, the term “steviol glycoside(s)” refers to glycosides of steviol. Exemplary steviol glycoside, include, but not are not limited to, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside 0, stevioside, steviolbioside, dulcoside A, rubusoside. Engineered yeast can produce steviol glycosides that are the same as steviol glycosides found in nature (“naturally occurring”) as well as steviol glycosides that are not found in nature. Steviol glycosides can be formed in an engineered yeast by enzymatic processes.

Structurally, steviol glycosides have a central molecular moiety, which is a single steviol base, and glucopyranosyl residues attached to the C13 and/or C19 atoms of the steviol base, according to the atom numbering on the base shown below. That is, glucopyranosyl residues represent groups R₂ and R₁ in the following formula:

Table A below shows the various steviol glycosides and the corresponding R₁ and R₂ groups:

TABLE A Compound name R₁ (C-19) R₂ (C-13) Steviol H H Stevioside β-Glu β-Glu-β-Glu (2->1) Rebaudioside A β-Glu

Rebaudioside B H

Rebaudioside C β-Glu

Rebaudioside D β-Glu-β-Glu (2->1)

Rebaudioside E β-Glu-β-Glu (2->1) β-Glu-β-Glu (2->1) Rebaudioside G β-Glu β-Glu-β-Glu (3->1) Rebaudioside M

Rebaudioside N

Rebaudioside O

Glu: glucose Rha: rhamnose

According to the current disclosure, steviol glycosides are produced in a process having at least two phases: first and second phases where a glucose-containing feed composition is provided to the medium in different modes of feeding in each phase, such as variable feeding and then constant feeding. A two phase feeding process as described herein can result in a growth rate that is slower in the second phase than in the first phase, and consequently increased steviol glycoside production rates, reduced fermentation times, and reduced biomass concentrations. The engineered yeast can have a set of enzymes that provide a pathway for the synthesis of steviol glycosides. For example, the process can produce steviol glycosides such as RebM and RebD.

The method of the disclosure can use various yeast host cells engineered to provide a pathway to one or more steviol glycosides. Such cells can be transformed with one or more DNA construct(s) encoding enzymes for steviol glycoside synthesis. Exemplary yeast that can be used for hosts for exogenous DNA constructs encoding steviol glycoside pathway enzymes, include, but are not limited to species of Candida, Kloeckera (Hanseniaspora), Kluyveromyces, Lipomyces, Pichia (Hansenula), Rhodotorula, Saccharomycete, Saccharomyces, Schizosaccharomyces, Torulopsis, Torulaspora, Yarrowia, and Zygosaccharomyces. Exemplary species are Candida albicans, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pompe, and Yarrowia lipolytica. Further, host cells can also include genetic modifications other than those of the steviol glycoside pathway that may provide improved performance during fermentation.

An “engineered yeast” refers to yeast cells having at least one exogenous DNA sequence that is introduced into the cell, either integrated into the cell's genome or present on an extrachromosomal construct, such as a plasmid or episome. The term “exogenous” refers to a molecule, such as a nucleic acid, or an activity, such as an enzyme activity, that is introduced into the host yeast. An exogenous nucleic acid can be introduced into the yeast host by well-known techniques and can be maintained external to the hosts chromosomal material (e.g., maintained on a non-integrating vector), or can be integrated into the yeast's chromosome, such as by a recombination event. Generally, the genome of an engineered yeast is augmented through the stable introduction of one or more recombinant genes. An exogenous nucleic acid can encode an enzyme, or portion thereof, that is either homologous or heterologous to the yeast. An exogenous nucleic acid can be in the form of a “recombinant gene or DNA construct” referring to a nucleic acid that is in one or more ways manipulated through molecular techniques to be in a form that does not naturally exist.

The term “heterologous” (e.g., “non-native”) refers to a molecule or activity that is from a source that is different than the referenced molecule or organism. Accordingly, a gene or protein that is heterologous to a referenced organism is a gene or protein not found in that organism. In the context of the disclosure, a “heterologous glycosyltransferase” refers to a glycosyltransferase polypeptide that is different from any glycosyltransferase polypeptide that may be native to the host organism. For example, a specific glycosyltransferase gene found in a first species and exogenously introduced into a host yeast organism that is different than the first species is “heterologous” to the host yeast.

The engineered yeast can use an auxotrophic marker suitable for selecting for a transformant having a nucleic acid encoding a steviol glycoside pathway enzyme. The host yeast can include modifications (deletions, and the like) in one or more genes that control auxotrophies, such as LYS2, LEU2, HIS3, URA3, URA5, and TRP1. Using a host cell having a desired genetic background for introduction of one or more exogenous genes, one or more gene construct(s) is introduced into a cell to integrate into the genome, or to be stably maintained and allow for expression. Methods for introducing a gene construct into a host cell include transformation, transduction, transfection, co-transfection, and electroporation. In particular, yeast transformation can be carried out using the lithium acetate method, the protoplast method, and the like. The gene construct to be introduced may be incorporated into a chromosome in the form of a plasmid, or by insertion into the gene of a host, or through homologous recombination with the gene of a host. The transformed yeast into which the gene construct has been introduced can be selected with a selectable marker (for example, an auxotrophic marker as mentioned above). Further confirmation can be made by measuring the activity of the expressed protein, or the production of a bioproduct such as a steviol glycoside.

The transformation of exogenous nucleic acid sequences including the steviol pathway genes can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of the introduced nucleic acid sequences or their corresponding gene product. It is understood by those skilled in the art that the exogenous nucleic acid is expressed in a sufficient amount to produce the desired product, and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and as disclosed herein.

The terpenoid compounds isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) can serve as chemical precursors to steviol glycosides in an engineered yeast. Some organisms, including plants, insect, and some microbial species, have a mevalonate (MVA) pathway that converts acetyl-CoA through a series of chemical intermediates to IPP and DMAPP. Some organisms produce IPP and DMAPP through the non-mevalonate pathway (also known as the methyl D-erythritol 4-phosphate or MEP pathway) starting with glyceraldehyde-3-phosphate (G3P) and pyruvate (PYR).

The yeast Saccharomyces cerevisiae naturally expresses genes of the mevalonate pathway. Mevalonate pathway genes include: (a1) acetoacetyl CoA thiolase (EC 2.3.1.9), (b1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5); (c1) HMG-CoA reductase (EC 1.1.1.34); (d1) mevalonate kinase (EC 2.7.1.36); (e1) phosphomevalonate kinase (EC 2.7.4.2); and (f1) mevalonate diphosphate decarboxylase (EC 4.1.1.33). Enzymes of the mevalonate pathway converts acetyl-CoA to IPP as follows: acetyl-CoA→acetoacetyl-CoA→3-hydroxy-3-methylglutaryl-CoA→mevalonate→mevalonate-5-phosphate→mevalonate-5-pyrophosphate→IPP. See also FIG. 1.

In some embodiments, the engineered yeast can include one or more modifications to increase the flux from acetyl-CoA to IPP and/or DMAPP, thereby providing an increased pool of IPP and/or DMAPP for use in a pathway to steviol. The modifications can include, for example, increasing expression or activity of one or more mevalonate pathway enzymes (a1)-(f1), such as by placing a nucleic acid encoding an enzyme that is homologous or heterologous to the yeast cell under the control of a promoter that provides increased expression, using multiple copies of the nucleic acid, and/or using a heterologous enzyme, a variant enzyme (e.g., one including one or more amino acid substitutions), or a variant heterologous enzyme that provides a higher level of enzymatic activity as compared to the native enzyme.

Alternatively, the non-mevalonate (MEP) pathway can be used to provide IPP and DMAPP as precursors to steviol glycoside production. The yeast Saccharomyces cerevisiae do not naturally express genes of the MEP pathway, but can optionally be engineered to provide MEP pathway genes. Theoretically, the MEP pathway is more energetically efficient generally because it loses less carbon as CO2 as compared to the MVA pathway (MEP pathway: 1 CO2/IPP; MVA pathway: 4 CO2/IPP; sugar as carbon source).

In particular, in the non-mevalonate (MEP) pathway compounds isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP) are generated through a series of intermediates leading from glyceraldehydes-3-phosphate (G3P) and pyruvate (PYR), and a number of enzymes are responsible for this conversion. Enzymes involved in a biosynthetic pathway from G3P and PYR to IPP and DMAPP include (a2) 1-deoxy-D-xylulose-5-phosphate synthase (DXS), (b2) 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (ispC)-, (c2) 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD), (d2) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), (e2) 2C-Methyl-D-erythritol-2,4-cyclodiphosphate Synthase (IspF), (f2) 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG), (g2) 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH), and (h2) isopentenyl-diphosphate isomerase (IDI). See FIG. 2

The methods of the disclosure for producing steviol glycoside(s) by fermentation can use engineered yeast that have one or more genetic modifications to increase the flux from G3P and PYR to IPP and/or DMAPP, thereby providing an increased pool of IPP and/or DMAPP for use in a pathway to steviol. The modifications can include, for example, increasing expression or activity of one or more enzymes (a2)-(h2), such as by placing a nucleic acid encoding an enzyme that is heterologous to the yeast cell under the control of a promoter that provides increased expression, using multiple copies of the nucleic acid, and/or using a heterologous enzyme, a variant enzyme (e.g., one including one or more amino acid substitutions), or a variant heterologous enzyme that provides a high levels of enzymatic activity.

The methods of the disclosure for producing steviol glycoside(s) by fermentation can use engineered yeast can also include a pathway to convert IPP and/or DMAPP to steviol. For example, in some aspects the engineered yeast can include exogenous nucleic acids expressing the following enzymes: (a3) geranyl geranyldiphosphate synthase (GGPPS), (b3) copalyl diphosphate synthase (CPS), (c3) kaurene synthase (KS), (d3) kaurene oxidase (KO), and (e3) kaurenoic acid 13-hydroxylase (KAH). See FIG. 3 Enzymes of the mevalonate pathway converts IPP and/or DMAPP to steviol as follows: IPP/DMAPP→geranyl geranyldiphosphate→copalyl diphosphate→kaurene→kaurenoic acid→steviol. See FIG. 3 Exogenous nucleic acids encoding enzymes (a3)-(e3) that are heterologous to the yeast cell can be placed under the control of a promoter that provides increased expression, using multiple copies of the nucleic acid, and/or using a variant enzyme (e.g., one including one or more amino acid substitutions), or a variant heterologous enzyme that provides a high levels of enzymatic activity.

The methods of the disclosure for producing steviol glycoside(s) by fermentation can use engineered yeast having any pathway to convert steviol to a steviol glycoside. If more than one steviol glycoside pathway enzymes are present in the engineered yeast, the yeast may be able to produce different steviol glycosides. For example, the yeast may be able to produce two, three, four, five, six, seven, eight, nine, ten, or more than ten different steviol glycoside species.

The steviol glycoside pathway can include one or more uridine diphosphate (UDP) glycosyltransferases (UGTs) that mediate the transfer of glycosyl residues from activated nucleotide sugars to acceptor molecules. In the case of a steviol glycoside pathway, a monosaccharide unit can be transferred to a hydroxyl or carboxyl moiety on a steviol or steviol glycoside molecule, or to a hydroxyl group on a glucose group that is attached to the steviol base. See FIG. 4. UGTs have been classified into families and subfamilies based on sequence homology. See Li, et al., 2001, J. Biol. Chem. 276:4338-4343. A superfamily of over 100 genes encoding UGTs, each containing a 42 amino acid consensus sequence, has been identified in the model plant Arabidopsis thaliana, and genes encoding UGTs have also been identified in several other higher plant species.

Exemplary UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to the steviol and or steviol glycoside substrate to provide the target steviol glycoside. In one embodiment, the engineered yeast can include one or more UDP-glucosyltransferase selected from group UGT74G1 (SEQ ID NO: 1), UGT85C2 (SEQ ID NO: 2), UGT76G1 (SEQ ID NO: 3), UGT91D2 (SEQ ID NO: 4), and also UGTs having substantial identity (e.g., >85%, >75%, >65%, >55%, >45% and >35%) to these polypeptides. An engineered yeast can include one or more exogenous nucleic acid molecule(s) that code for these UGTs.

The engineered yeast can also include one or more UGT and UDP-glucose recycling enzyme(s). An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside is UGT91D2. An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A is UGT76G1. An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D is UGT91D2. An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M is UGT76G1.

Exemplary publications that describe engineered microorganisms for steviol glycoside production and steviol glycoside pathway enzymes include, for example, US2014/0357588, WO2014/193934, WO2014/193888, and WO2014/122227, each of which are hereby incorporated by reference in their entirety.

In one embodiment, an engineered yeast useful for the production of steviol glycosides expresses the following enzymes: geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl diphosphate synthase (CDPS), kaurene oxidase (KO), kaurene synthase (KS); steviol synthase (KAH), cytochrome P450 reductase (CPR), UGT74G1, UGT76G1, UGT91D2, UGT85C2 and a EUGT11. WO2014/122227 describes an engineered yeast strain that express these enzymes. The UDP-glucosyltransferases can be a gene encoding a polypeptide for example, UGT74G1 (SEQ ID NO: 1), UGT85C2 (SEQ ID NO: 2), UGT76G1 (SEQ ID NO: 3), UGT91D2 (SEQ ID NO: 4), and a EUGT11 (SEQ ID NO: 13); these genes encode polypeptides capable of carrying out a number of reactions such as a) a gene encoding a polypeptide capable of beta 1,2 glucosylation of the C2′ of the 19-O glucose of a steviol glycoside; (b) a gene encoding a polypeptide capable of beta 1,2 glucosylation of the C2′ of the 13-O-glucose of a steviol glycoside; (c) a gene encoding a polypeptide capable of beta 1,3 glucosylation of the C3′ of the 19-O-glucose of a steviol glycoside; (d) a gene encoding a polypeptide capable of beta 1,3 glucosylation of the C3′ of the 13-O-glucose of a steviol glycoside; (i) a gene encoding a polypeptide capable of glucosylation of the 13-OH of steviol or a steviol glycoside; (j) a gene encoding a polypeptide capable of glucosylation of the C-19 carboxyl of steviol or a steviol glycoside. For example, UGT85C2 carries out reaction (i); UGT74G1 carries out reaction (j); UGT91D2 carries out reactions (a; weakly), (b); UGT76G1 carries out reactions (c) and (d) EUGT11 carries out reactions (a), (b; less well).

The term “medium” refers to a liquid composition in which the engineered yeast or fungus can be maintained, can grow, can ferment, or combinations thereof. A “medium” may also be referred to as a “broth” or “cell culture,” and terms such as “growth,” “division,” “respiration,” and “fermentation” may be used to more specifically define the type of cellular activity that is occurring in the medium.

A medium can be defined with regards to the components present in the medium, and amounts thereof, such as (a) carbon sources, including carbohydrates such as glucose and starch products such as maltodextrin; (b) nitrogen sources, such as yeast nitrogen base, ammonium hydroxide, urea, ammonium sulfate, or any combination thereof; (c) salts, such as potassium phosphate (monobasic, dibasic), magnesium sulfate, sodium chloride, and calcium chloride; (d) vitamins, such as biotin, calcium pantothenate, folic acid, (myo)-inositol, nicotinic acid, p-aminobenzoic acid, pyridoxine HCl, riboflavin, thiamine HCL, and citric acid; and/or (e) trace metals such as boric acid, copper sulfate, cobalt chloride, calcium chloride, potassium iodide, ferric chloride, magnesium sulfate, manganese chloride, sodium molybdate, and zinc sulfate. Components in the medium can be defined on a dry weight basis. Further, the medium is water-based, or an “aqueous” composition. The medium can also be defined with regards to its pH, and biocompatible acids, bases, and buffers that are used to control the pH in the medium.

In exemplary embodiments, the concentration of glucose in the medium in steps (a) and (b) is kept in the range of about 0 g/L to about 5 g/L, or 0 g/L to about 2 g/L. In exemplary embodiments, the concentration of a nitrogen source (total amount) in the medium, such as yeast nitrogen base, ammonium hydroxide, urea, ammonium sulfate, yeast extract is in the range of about 5 g/L to about 40 g/L. In exemplary embodiments, the concentration of salts (total amount) in the medium, such as salts including magnesium sulfate in the range of about 0 g/L to about 12 g/L, and potassium phosphate in the range of about 0 g/L to about 22 g/L. In exemplary embodiments, the concentration of trace metals (total amount) in the medium is kept in the range of about 0 g/L to about 0.4 g/L, or 0 g/L to about 0.2 g/L.

A composition (a “feed composition”) can be added to the medium that includes the engineered yeast to increase the volume of the medium, and as the engineered yeast grows in the medium, the amount of biomass. The feed composition can include components for yeast growth and fermentation to form a desired medium. The feed composition can include carbohydrate(s), a nitrogen source, such as ammonium hydroxide, urea, ammonium sulfate, yeast extract, or any combination thereof; salts, vitamins, and trace metals. The concentration of the components in the feed composition may be greater than the concentration of components in the medium so that when the feed composition is added it provides desired amounts of components in the medium suitable for fermentation of the engineered yeast.

Fermentation of the engineered yeast can be performed using starch and/or sugar containing plant material derivable from any plant and plant part, such as tubers, roots, stems, leaves and seeds. Starch and/or sugar-containing plant material can be obtained from cereal, such as barley, wheat, maize, rye, sorghum, millet, barley, potatoes, cassava, or rice, and any combination thereof. The starch- and/or sugar-containing plant material can be processed, such as by methods such as milling, malting, or partially malting. In some embodiments, the medium for steps (a) and (b) includes a treated starch. For example, the medium for growth and/or fermentation can include a partially hydrolyzed starch. The partially hydrolyzed starch can include high molecular weight dextrins and high molecular weight maltodextrins. A partially hydrolyzed starch product can be used that has amounts of starch and starch degradation products within desired ranges beneficial for steviol glycoside production.

Optionally, a starch degrading enzyme can be added to the medium that includes a starch material in order to increase the concentration of monomeric sugars such as glucose that can be utilized by the engineered yeast during the fermentation stage. Exemplary starch-degrading enzymes include amylolytic enzymes such as glycoamylase and amylase. In some embodiments, fermentable sugars such as fructose, sucrose, maltose, maltotriose, and the like can be included in the medium instead of or in addition to glucose.

In some optional modes of practice, fermentation can be carried out in medium that includes steviol-containing compounds. Such compounds can be directly used by the glucosyltransferases in the engineered yeast. For example, optionally, fermentation can be carried out in medium containing steviol-13-O-glucoside or steviol-19-O-glucoside. Using this medium, the microorganism may contain and express genes encoding a functional EUGT11, a functional UGT74G1, a functional UGT85C2, a functional UGT76G1, and a functional UGT91 D2. Compounds such as rebaudioside A, rebaudioside D, and rebaudioside M may be obtained from the fermentation medium. As another option, fermentation can be carried out in medium containing rubusoside. Using this medium, the microorganism may contain and express genes encoding a functional EUGT11, a functional UGT76G1, and a functional UGT91D2. Compounds such as rebaudioside A, D, and M may be obtained from the medium following fermentation.

In some cases fermentation is carried out in industrial capacity fermenters in order to achieve commercial scale economic benefits and control. In an embodiment, the fermentation is carried out in a fermenter that has a capacity of about 10,000 liters or more.

The terms “first phase” and “second phase” (and optionally, “pre-phase,” “third phase,” “fourth phase,” fifth phase,” etc., if necessary) may be used to describe aspects of the method of producing steviol glycosides with regards to the medium. The term “stage” may also be used for “phase.” The process includes two or more phases where the medium is treated differently in each phase, such as by adding a feed composition to the medium in a second, later, phase of the process in a mode that is different than a mode of adding the feed composition in the first, earlier, phase. The difference in mode of addition affects the growth of the engineered yeast, and production of the steviol glycosides during the process.

Prior to the first phase (in which cell growth is controlled by the first mode of adding), the cells can be cultured according a “pre-phase.” The pre-phase can be a “seed/initial growth phase” in which cells are grown in a medium to become acclimated to the medium components (carbohydrates, nitrogen source, salts, vitamins, trace metals). In the pre-phase carbohydrate supply to the cells is not modulated as it is during the first and second phases, so the cells may grow at their maximum biological rate. For example, the cells in the pre-phase may be batch fed. As the cells become acclimated to the medium, the cells will enter a growth phase and increase in cell numbers. During the pre-phase, the engineered yeast can multiply by budding, referred to as yeast division.

For example, during the pre-phase a growth composition that includes carbohydrate(s), a nitrogen source, such as yeast nitrogen base, ammonium hydroxide, urea, ammonium sulfate, or any combination thereof, salts, vitamins, and trace metals can be added to medium that includes the engineered yeast in a batch process. In some modes of practice a composition is added to provide a medium that has ammonium hydroxide, urea, ammonium sulfate, or combinations thereof, as the sole nitrogen source. The same composition can be used as a feed composition in the subsequent first phase, where cell growth is controlled by the mode of addition of the feed composition to the medium.

Following the pre-phase, which is characterized by rapid cell growth and increase in biomass, the first phase (e.g., step a) can be commenced by regulating addition of the glucose containing composition according to the first mode of adding. The first phase can be described in various ways, such as by the how the feed solution is added to the medium and how the cells grow in response to that type of addition.

The mode of addition can affect the doubling times of the engineered yeast. The doubling times in the first phase can be greater (slower growth) than the doubling times in the pre-phase. During the first phase the biomass of the medium can increase, but it may increase at a rate that is lower than the increase seen in the pre-phase. The first phase can also be described in terms of how the cells grow as compared to the second phase, where feed solution is added to the medium in a second mode that is different than the first mode.

For example, in the first phase the yeast can be grown in a medium under conditions to achieve one or more growth rate(s). The growth rates may be controlled by controlling the feed rate of the feed medium to the fermenter to reach certain dilution rate(s)) that are within a first range that is greater than growth in the second phase. For example, in the seed/growth phase the growth rate (μ) can be about 0.06 h⁻¹ or greater, such as a rate in the range of about 0.06 h⁻¹ to about 0.17 h⁻¹, or about 0.09 h⁻¹ to about 0.15 h⁻¹. Growth can be measured by optical density, for example at 600 nm. Growth rate can be controlled by adjusting the feed rate of the feed medium based on growth rate measurements, for example by increasing the feed rate to increase growth rate or by decreasing feed rate to decrease growth rate.

Optionally, the first phase can be described in terms of glucose concentration in the medium. For example, in some modes of practice, the first phase is started at a time when there is less than 3 g/L of glucose in the medium (glucose may be determined by using YS12700 Select Chemical Analyzer, Yellow Springs, Ohio). For example, the amount of glucose in the medium during the pre-phase can be monitored and when the concentration drops below 3 g/L, the first phase feeding can be started.

A desired growth rate in the first phase can be achieved by adding a composition comprising glucose to the medium according to a first mode. A “mode of feeding” refers to a way a feed composition that includes glucose is added to the medium having the engineered yeast. Modes of feeding include constant rates of feeding, non-constant rates of feeding, continuous addition of feed composition, bulk addition of feed composition, etc. In some modes of feeding, a feed composition is added to the medium at a non-constant rate of feeding during the first phase. For example, the non-constant rate of feeding can be a variable rate of feeding.

A variable rate of feeding refers to adding a feed solution to the medium at two or more different rates over a period of adding a feed solution to the medium. In some modes of practice, during a variable rate feeding, the rate decreases over a period of time. For example, in a growth phase of the process the feeding can change from a higher rate of feeding earlier in the growth phase to a lower rate of feeding later in the growth phase. This can be carried out by constantly decreasing rate of feeding, or can be carried out by a series of small decremental steps. In an optional mode of practice, a variable rate of feeding can include increasing the rate of feeding and then decreasing the rate of feeding.

A variable rate of feeding can be achieved using a variable rate addition system. Examples of such systems include a variable speed pump or a metering valve (such as a throttle valve) operably connected to a pump, which pump or valve can be utilized to vary the amount of feed composition introduced into the fermentation medium over time.

The first phase may also be explained with reference to one or more parameters associated with the medium, such as the period of time of the first phase, the temperature of the medium, the amount of biomass grown, and the pH of the medium. In some modes of practice, the first phase with a variable rate of feeding can be carried out for a period of time of about two hours or greater and up to about 40 hours. For example, the first phase can be about 10 hours or greater, such as a period of time in the range of about 10 hours to about 30 hours, or about 10 hours to about 24 hours. The first phase may encompass all or part of the lag phase of growth, and all or part of the log (exponential) phase of growth of the engineered yeast. After this period of time the mode of adding the feed composition including glucose to the medium can then be changed (e.g., to a constant rate of feeding in the second phase).

In exemplary modes of practice, in the first phase the medium is kept at a temperature in the range of about 25-35° C., or 28-32° C., and most preferably at about 30° C. Also, growth of the engineered yeast can be performed with aeration, and/or with agitation. Aeration conditions can have an effect on the amount of oxygen dissolved in the medium, and therefore the oxygen available to the engineered yeast. The amount of oxygen uptake by the engineered yeast can be controlled by the rate at which oxygen is supplied the formation of small oxygen bubbles in the medium, which can be achieved through agitation and/or sparging.

In the medium and during the first phase, the aeration can be performed. Aeration may be described in terms of dissolved oxygen transfer rate to the medium in units of mg min⁻¹ liter⁻¹. Aeration may also be described in terms of the dissolved oxygen (%). (For example, see Anderlei, T., and Büchs, J. (2000) Biochem. Engin. J. 3478:1-6). A sparging technique that promotes the formation of fine gas bubbles can be performed to provide desired aeration. In some modes of practice, during the first phase, agitation and aeration are increased, such as in a stepwise manner. Methods of the disclosure using a two phase feeding process can also reduce the aeration needs in the medium while still providing desired steviol glycoside production. In some modes of practice the dissolved oxygen is maintained at greater than 15%.

As used herein “biomass” refers to the weight of the engineered yeast, which can be measured in grams of dried cell weight per liter of medium (DCW/L). As another exemplary parameter, in some modes of practice, the first phase with a variable rate of feeding produces an amount of biomass of at least about 5 dcw/L. Preferably, the amount of biomass produced is in the in the range of about 5 g dcw/L to about 60 g dcw/L, about 20 g dcw/L to about 60 g dcw/L, or about 20 g dcw/L to about 40 g dcw/L.

As another example, in some modes of practice, the first phase with a variable rate of feeding is carried out at a pH of 6.0 or less, less than about 5.5, and preferably less than 5.2, such as in the range of about 4.0 to about 5.2. During the first phase the pH can be monitored to so that it stays within a desired, lower pH range, such as in the range of about 4.0 to 5.2. Acid or base can be added to the medium during the feeding to maintain the pH within a desired range.

After the first phase, the engineered yeast can enter the second phase, such as a “fermentation phase” where the mode of providing the feed composition is different than in the first phase. In the second phase the growth of the engineered yeast has at least slowed and are actively assimilating carbohydrate and producing steviol glycoside(s). As used herein “fermentation” is used to describe the phase of significant production of steviol glycoside(s), which can occur in fully aerobic, partially aerobic or anaerobic conditions. In partially aerobic conditions, both fermentative and respiratory pathways can be active, and some cell growth may occur. In partially aerobic conditions the amount of oxygen consumed can be less than during the seed/growth phase.

In the second phase, a feed composition with glucose can be added to the medium in a different mode than in the first phase. In some modes of practice, the first and second phases are carried out in the same vessel, wherein during the first phase a feed solution that includes glucose is added to the medium in the vessel at a variable rate, and then in the second phase the feed solution is added to the medium in the same vessel but at a constant rate.

In some modes of practice, in the second phase the feed composition is added to the medium at a constant feeding rate. For example, the constant rate of feeding is not greater than 10 g glucose/L media/h, and preferably at a constant rate of feeding in the range of 2 g glucose/L media/h to 10 g glucose/L media/h.

For example, in the second phase which includes fermentation and production of the steviol glycosides, the yeast can be grown in a medium under conditions to achieve one or more growth rate(s) that are within a range. For example, in the second phase the growth rate(s) can be about 0.09 h⁻¹ or less, such as a rate in the range of about 0.015 h⁻¹ to about 0.09 h⁻¹, or about 0.015 h⁻¹ to about 0.06 h⁻¹.

In some modes of practice, in the second phase with a constant rate can be carried out for a period of time to provide desired production of steviol glycosides. For example, the second phase can be started at a time of about 30 hours or later from the start of step (a), and then can be performed up to 130 hours from an from the start of step (a). The second phase may encompass all or part of the fermentation phase where the majority of steviol glycosides are produced. Preferably most of the steviol glycoside(s) (i.e., greater than 50%) are produced by the engineered yeast during the second phase. Methods of the disclosure including the two phase feeding provide a benefit with regards to fermentation, allowing up to about a 25% reduction, or even up to a 40% reduction in fermentation times as compared to a control process (e.g., a single phase fermentation).

Further, in some modes of practice, in the second phase with a constant rate of feeding can be controlled so the engineered yeast do not grow to a biomass amount of greater than 180 g dcw/L. Methods of the disclosure including the two phase feeding provide a benefit with regards to biomass production, allowing up to about a 25% reduction in the amount of biomass produced as compared to a control process with a single phase fermentation.

Further, in some modes of practice, during the second phase the medium can have a higher pH than the pH in the medium during the first phase. For example, at the start of, or during the second phase, a base can be added to the medium to increase the pH from a lower to a higher pH. The base can be present in the feed composition, or can be added separate from the feed composition for the second phase. For example, in the second phase the pH can be adjusted to about pH 5.8 or greater, or about pH 6.0 or greater, such as in the range of about pH 5.8 to about pH 7.5 or greater, or about pH 6.0 to about pH 7.0. During the second phase, the pH can be monitored (e.g., periodically or continuously) and adjustments to the medium can be made if the pH falls outside a desired range. For example, ammonium hydroxide can be added to the second medium if the pH drops below 6.0 or 5.8, so as to adjust the pH to about 6.0 or greater.

In exemplary modes of practice, fermentation and optionally growth in the second phase is performed at a temperature in the range of about 25-35° C., or 28-32° C., and most preferably at about 30° C. Also, fermentation and optionally growth of the engineered yeast in the second phase can be performed with aeration, and with agitation. Methods of the disclosure using a two phase feeding process can also reduce the aeration needs in the medium while still providing desired steviol glycoside production.

During fermentation, the medium can be monitored for the production of steviol glycosides. Fermentation can be stopped at a point where there is a desired steviol glycoside total amount and profile.

In some modes of practice, glucose feed rates of a fermentation producing steviol glycoside can be controlled based on variables such as Respiratory Quotient (RQ), oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) or combinations thereof. These variables can be measured in the broth or off gas. Controlling the glucose feed rate by these variables (e.g., Respiratory Quotient (RQ)) can increase the production, can increase the yield, decrease biomass production and decrease ethanol production of the desired steviol glycosides such as rebaudiosides D and rebaudioside M. Controlling the glucose feed rates can also increase the consistency of the fermentation operation, namely reducing failure rates of batches and reducing overall system variability due to glucose feed rate or culture physiology.

RQ can be used to control glucose feed rate to prevent the toxic ethanol accumulation, a by-product of fermentative metabolism.

RQ is defined as the molar rate of carbon dioxide produced divided by the molar rate of oxygen consumed in the culture. RQ can be measured by analyzing the exhaust gas coming from the fermentor for content of carbon dioxide and oxygen. This metabolic parameter can be measured continuously or intermittently throughout the desired production phase. In some modes of practice, appropriate intervals for measurements are every four hours, two hours, hourly, half-hour, quarter-hour, ten minutes, five minutes, four minutes, three minutes, two minutes, or one minute. Time periods during measurements may vary with growth conditions, from initiating the culture through production of steviol glycosides. Exemplary periods for measurement and control are between 20 and 40 hours, between 10 and 60 hours, between 5 and 70 hours, and between 20 and 110 hours after initiating of the culturing in the fermentor.

In the presence of oxygen, yeast cells use aerobic metabolism, which is more efficient, e.g., more energy is obtained from a mole of glucose under aerobic metabolism than under fermentative metabolism.

The RQ of a medium producing only ethanol from glucose approaches infinity (since little or no oxygen is consumed, the denominator of RQ approaches zero), whereas for purely aerobic metabolism of glucose the RQ approaches the value of 1.0 (three moles of oxygen are consumed to produce 3 moles of carbon dioxide). Thus, values higher than 1 indicate a mixed metabolic condition where both aerobic and fermentative metabolism are taking place simultaneously. Typically, oxygen transfer rate and/or glucose feed rate (or the rate of feeding other carbohydrate(s)) can be adjusted using RQ as a feedback control variable to accomplish this mixed metabolism.

RQ can be measured in the exhaust gas stream from a fermentor. Any known and suitable method for ascertaining the molar concentration of oxygen consumed and carbon dioxide generated can be used. Exemplary techniques which may be used are mass spectrometry, infrared spectroscopy, and paramagnetic analysis. Exemplary software that may be used, for example with a mass spectrophotometer, include GasWorks from Thermo Scientific™.

In some embodiments, the RQ is maintained at about 0.5 to about 2.0. In some modes of practice, the RQ is maintained from about 0.9 to about 1.5, or about 1.0 to about 1.3. Maintaining the RQ in the disclosed ranges can result in improved steviol glycoside production. For example, some modes of practice result in improved Reb D and Reb M production.

When RQ is maintained in a narrow range from approximately 1.1 to approximately 2, ethanol accumulation stabilizes at levels that are not toxic. In some embodiments, the concentration of ethanol is maintained between about 5 g/L and 17 g/L. RQ ranges that may be desirable include about 1.08-2.0; about 1.08-1.85; about 1.08-1.65; about 1.08-1.45; about 1.08-1.35; about 1.08-1.25; about 1.08-1.2; and about 1.08-1.15. Other suitable RQ ranges include 1.08 to 1.35, and 1.15 to 1.25. In some embodiments, the glucose addition rate is adjusted to maintain the RQ in the about from 0.5 to about 2.0, from 0.9 to about 1.5, or from 1.0 to about 1.3.

RQ can be monitored and controlled during any desired portion of the fermentation, for example from 0 to 110 hours, from 20-40 hours, from 20-70 hours, from 20-90 hours, from 20-110 hours, or any other desired time period. In some embodiments, the RQ is monitored during phase II feeding or fermentation phase.

Thus, RQ can be manipulated and changed over time by addition of various carbon sources, by addition of various amounts of a carbon source, and by manipulation of the oxygen levels. In one embodiment, oxygen levels are manipulated by increasing or decreasing agitation. In another embodiment, the ratio of oxygen to nitrogen gas in a gas feed is controlled. Ways that the oxygen transfer rate can be adjusted include changing the air flow rate, the oxygen concentration, the cell density, the temperature, and/or agitation. In some embodiments, glucose or other fermentable sugar feed is modulated to affect the RQ. Other fermentable sugars which can be used in the feed include without limitation fructose, sucrose, maltose, and maltotriose. Feed rate or composition can be modulated to affect the RQ. The control of RQ may be manual or automatic.

The “total steviol glycosides” refers all the steviol glycosides present in the medium after a period of fermentation, which includes the amount of steviol glycosides in the liquid medium and obtainable from the engineered yeast. The steviol glycoside content can be expressed with regards to a total steviol glycosides amount in the medium, or the amount of one or more, but not all, steviol glycosides, in the medium. The amount of steviol glycosides in the composition can be expressed in relation to one another, or to the total amount of steviol glycosides, such as by a weight percentage of the total amount of steviol glycosides, or a ratio, or range of ratios, expressed as weight percent, or molar percent. The amount of steviol glycosides can also be expressed relative to a control sample, such as a control sample prepared by a process that does not include the first and second stages of feeding.

In some modes of practice, method of the disclosure provides improvement in the production of certain steviol glycosides, such as rebaudioside D and rebaudioside M. In some embodiments, a combined production rate of rebaudioside D and rebaudioside M is at least 0.02 g/L/h, 0.03 g/L/h, 0.04 g/L/h, 0.05, 0.06, 0.07, or 0.075 g L⁻¹ h⁻¹.

Methods of the disclosure can provide an improvement in the rate of steviol glycoside production during fermentation. For example, engineered yeast that are grown and fermented using the first and second phase method as described herein can exhibit an increase in the rate of steviol glycoside production that is about 1% or greater, about 2% or greater, about 3% or greater, about 5% or greater, about 7% or greater, about 10% or greater, about 12% or greater, or about 15% or greater, relative to the rate of steviol glycoside produced via an engineered yeast strain that is grown and fermented in a control process (1^(st) phase μ=0.12 h⁻¹; 2^(nd) phase 7.71 g glucose L⁻¹ h⁻¹).

The phased feeding according to the disclosure can result in Reb D and Reb M production and increased production rates, increased yields, reduced fermentation times and reduced biomass concentrations.

Following the second phase wherein fermentation produces steviol glycoside(s), a composition containing one or more steviol glycoside(s) can be obtained from the medium using various techniques. In some embodiments, a compound such as permeabilizing agent can be added to the medium to enhance removal of the steviol glycosides from the cell and into the medium.

The medium can then be centrifuged or filtered to remove the engineered cells. The medium can optionally be treated to remove low molecular weight components (glucose, basic nutrients, and salts), such as by membrane dialysis. Depending on a desired use, a composition comprising one or more steviol glycoside compound(s) can be used.

After fermentation the engineered yeast can optionally be treated using a heat treatment method to enhance the recovery of steviol glycosides. After fermentation, but before any heat, treatment the medium may contain a suboptimal amount of the steviol glycosides, with the most of the desired steviol glycosides within the engineered yeast. To increase the recovery of steviol glycosides, in some modes of practice a composition, such as the medium at the higher pH in which the engineered yeast have been fermented, is heated to a temperature in the range from 50° C. to 95° C., or 70° C. to 95° C., for a period of time in the range of 5 minutes to 48 hours.

If it is desired to provide a composition with steviol glycosides in enriched or purified form, or where certain steviol glycosides are separated from one another, further purification can be carried out. Such enrichment or purification of steviol glycoside components can be carried out on the medium in which fermentation took place, or the medium can then be dried down prior to purification. For example, medium can be dried down using lyophilization to form a dry composition (e.g., powder or flakes) including steviol glycosides that can be subsequently processed.

As used herein, the term “total steviol glycosides” (TSG) is calculated as the sum of the content of all steviol glycosides in a composition on a dry (anhydrous) basis.

In some modes of practice, dried fermentation broth enriched for steviol glycosides is used as the starting material for purification. For example, a solvent or solvent combination can be added to the dried fermentation broth to dissolve or suspend material that includes the steviol glycosides. An exemplary combination for dissolving the steviol glycosides is a mixture of water and an alcohol (e.g., 50:50 ethanol:water). To facilitate dissolving or suspending, the dried broth materials can be heated at a temperature above room temperature, such as in the range of 40° C.-60° C. Mechanical disruption of the dried broth materials can also be performed, such as by sonication. The dissolved or suspended broth materials can be filtered using a micron or sub-micron prior to further purification, such as by preparative chromatography.

Dried fermentation broth enriched for steviol glycoside compounds can be subjected to purification, such as by reverse phase liquid chromatography. A suitable resin can be used to retain steviol glycoside compounds in the column, with removal of hydrophilic compounds which get washed through the column with a liquid such as water. Elution of steviol glycosides from the column can be accomplished a suitable solvent or solvent combination such as acetonitrile or methanol.

Elution of steviol glycosides from a reverse phase column can yield a composition which can be useful for any one of a variety of purposes. For example, a purified steviol glycoside composition can be used as a sweetener composition for oral ingestion or oral use. The composition can be defined with regards to the steviol glycosides in the composition.

Steviol glycoside-producing S. cerevisiae strains were constructed using methods as described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in their entirety. The following sequences were used for construction of a parent strain (Strain A): a recombinant gene encoding a Synechococcus sp GGPPS polypeptide (SEQ ID NO:6), a recombinant gene encoding a truncated Zea mays CDPS polypeptide (SEQ ID NO:7), a recombinant gene encoding an Arabidopsis thaliana KS polypeptide (SEQ ID NO:8), a recombinant gene encoding a recombinant Stevia rebaudiana KO polypeptide (SEQ ID NO:9, SEQ ID NO:10), a recombinant gene encoding an Arabidopsis thaliana ATR2 polypeptide (SEQ ID NO:11, SEQ ID NO:12), a recombinant gene encoding an Oryza sativa EUGT 11 polypeptide (SEQ ID NO:13), a recombinant gene encoding an SrKAHe1 polypeptide (SEQ ID NO:14, SEQ ID NO:15), a recombinant gene encoding an Stevia rebaudiana CPR8 polypeptide (SEQ ID NO:16, SEQ ID NO:17), a recombinant gene encoding an Stevia rebaudiana UGT85C2 polypeptide (SEQ ID NO:2), a recombinant gene encoding an Stevia rebaudiana UGT74G1 polypeptide (SEQ ID NO:1), a recombinant gene encoding an Stevia rebaudiana UGT76G1 polypeptide (SEQ ID NO:3), and a recombinant gene encoding an Stevia rebaudiana UGT91D2 variant (or functional homolog), UGT91D2e-b, (SEQ ID NO:4) polypeptide produced steviol glycosides.

The UGT91D2e-b variant of UGT91D2 (SEQ ID NO:5 from PCT/US2012/050021) includes a substitution of a methionine for leucine at position 211 and a substitution of an alanine for valine at position 286. (Additional variants, except T144S, M152L, L213F, S364P, and G384C variants, described in Table 12 and Example 11 of PCT/US2012/050021, which is hereby incorporated by reference in its entirety, could be used.) GeneArt codon-optimized sequence encoding a Stevia rebaudiana UGT91D2e-b with the amino acid modifications 1,211M and V286A (SEQ ID NO:4 for amino acid sequence; codon optimized nucleotide sequence is set forth in SEQ ID NO:5).

Strain B is derived from the parent strain described above and additionally includes a codon-optimized CPR1 from Stevia rebaudiana (SEQ ID NO:18 corresponding to amino acid SEQ ID NO:19).

Example 1 Production of Reb D and Reb M in a Two-Phase Feeding Process

For inoculum preparation, the yeast strain B was cultured in 150 mL of seed flask medium in 1 liter shake flasks at 250 rpm and 30° C. for 20-24 hours.

TABLE 1 Seed Flask Medium Component Formula Concentration Units Biospringer 0251 yeast extract 7.5 g/L Glucose monohydrate C₆H₁₂O₆* H₂O 22.0 g/L

For the fermentation, 75 mL of seed culture was transferred into initial fermentation medium, as in Table 2, with an initial volume of 0.75 liters (38.5% of tank level). Fed batch fermentations were carried out in 2 L New Brunswick BioFlo310 fermenters. Fermentation was controlled at pH 5.0 with 12% NH₄O H and temperature was maintained at 30° C. throughout. The air flow rate was 1.75 SLPM and agitation rate was 1200 rpm throughout the fermentation.

Glucose concentration was kept limited by controlling flow rates of fermentation feed medium. A 2-phase feeding strategy involved an initial exponential phase (feed phase I) beginning at 12 hours (after inoculating the fermenter) with a growth rate of u=0.12 h⁻¹ or higher while the feed phase II started in the range of 35-39 hours with constant flow rates. The phase II feeding involved constant feeding in the range of 14.4 to 22.96 g glucose/L broth/h. Feeding was continued until 1.0 liter of fermentation feed medium was delivered. Antifoam, Ivanhoe 1163B, was added to the feed medium at 1.3 g/L and additional bolus additions of 5 wt % antifoam solution were added as needed.

The medium was based on Verduyn et al (Verduyn C, Postma E, Scheffers W A, and Van Dijken J P. Yeast. 1992 July; 8(7):501-17) with modifications as described in tables 2 and 3.

TABLE 2 Component Formula Concentration Units Initial Fermentation Medium Glucose monohydrate C₆H₁₂O₆* H₂O 22.0 g/L Ammonium sulfate (NH₄)₂SO₄ 5.0 g/L Monobasic potassium KH₂PO₄ 3.0 g/L phosphate Magnesium sulfate MgSO₄* 7 H₂O 0.5 g/L heptahydrate Trace metals stock 10.0 ml/L Vitamin stock 12.0 ml/L Trace Metals Stock Solution Disodium edetate C₁₀H₁₄N₂Na₂O₈ * 15 g/L 2H₂O Zinc sulfate ZnSO₄ * 7H₂O 4.5 g/L heptahydrate Manganese (II) MnCl₂ * 4H₂O 1.026 g/L chloride tetrahydrate Cobalt (II) chloride CoCl₂ * 6H₂O 0.32 g/L hexahydrate Copper (II) sulfate CuSO₄ * 5H₂O 0.3 g/L heptahydrate Sodium molybdate Na₂MoO₄ * 2H₂O 0.4 g/L dihydrate Calcium chloride CaCl₂ * 2H₂O 3 g/L dihydrate Iron (II) sulfate FeSO₄ * 7H₂O 3 g/L heptahydrate Boric acid H₃BO₃ 1 g/L Potassium iodide KI 0.1 g/L Vitamin Stock Solution d-Biotin C₁₀H₁₆N₂O₃S 50 mg/L Calcium pantothenate C₁₈H₃₂CaN₂O₁₀ 1000 mg/L Nicotinic acid C₆H₅NO₂ 1000 mg/L Thiamine hydrochloride C₁₂H₁₇ClN₄OS•HCl 1000 mg/L Pyridoxine hydrochloride C₈H₁₁NO₃•HCl 1000 mg/L p-aminobenzoic acid C₇H₇NO₂ 200 mg/L myo-inositol C₆H₁₂O₆ 25000 mg/L

TABLE 3 Fermentation Feed Medium Component Formula Concentration Units Glucose monohydrate C₆H₁₂O₆* H₂O 660 g/L Antifoam 1.3 g/L Potassium sulfate K₂SO₄ 4.2 g/L Sodium sulfate Na₂SO₄ 0.336 g/L Magnesium sulfate MgSO₄ * 7H₂O 6.12 g/L heptahydrate Monobasic potassium KH₂PO₄ 10.8 g/L phosphate Trace metal stock 14.4 mL/L Vitamin stock 14.4 mL/L

Quantification of steviol glycoside can be carried out by high performance liquid chromatography (HPLC) analysis as described below, and compared against calibration curves obtained using authentic standards purchased from Chromadex.

100 μL of the fermentation media were pipetted into a 2 mL microcentrifuge tube. 900 μL of 61% methanol (extraction solvent) was added into the 2 ml microcentrifuge tube and agitated by placing on a sample rotator for 10 min to extract the steviol glycosides. The samples were then centrifuged at 10K rpm in a microcentrifuge for 3 min and the clarified supernatant was pipetted into an autosampler vial for analysis.

UHPLC Method for Glycoside Separation

The steviol glycosides were separated using two Agilent SB-C18 RRHD columns in series (2.1 mm×150 mm, 1.8 um) with a stem filter assembly from Optimize Technologies installed as a pre-column filter. The mobile phase used was channel A: 0.01% trifluroacetic acid (TFA) in water and channel B acetonitrile. The flow rate was 0.38 mL/min, the column temperature was 65° C. and the detection was performed at ultraviolet absorption of 210 nm. The gradient elution profile is shown below:

Time % Channel A % Channel B 0 85 15 0.5 85 15 30 75 25 40 65 35 49 47 53 49.1 0 100 58 0 100 58.1 85 15 62 85 15

Calibration was performed using Reb A (98.85% purity) from Cargill, Inc lot 1008-005 in 55% MeOH at the following concentrations: 0.35, 0.175, 0.07, 0.035, 0.014, 0.007 mg/mL. All glycosides are quantitated off of the Reb A curve. Experimental correction factors for Reb D, Reb M, and Reb B were determined against Reb A while all other analytes are corrected by molecular weight.

Cell dry weight (biomass) is measured by filtering broth through a 0.45 micron filter and washing with 3 volumes of water and dried in a 105 oven for 18 hours.

TABLE 4 Increased glucose medium feed rates in both phase I and phase II feedings of the 2 phase feeding regime Phase II Reb DM Phase I feed rate Reb D Reb M Reb DM Reb DM Fermentation Biomass Yield feed rate g conc conc conc Rate RebD/rebM Time conc g/g (mu in h⁻¹) dx/L/h g/L g/L g/L mg/L/h ratio hours g/L Dx*100 0.12 7.71 1.08 1.89 2.97 25.2 0.57 117.9 114.4 0.96 0.15 10.5 0.85 1.67 2.52 27.8 0.5 90.75 111.4 0.82 0.18 12.3 0.49 0.99 1.48 19.5 0.49 75.8 90.3 0.48

Example 2

For inoculum preparation, the yeast strain B was cultured as described in Example 1 using the seed flask medium of Table 1, Example 1. The fermentation, the initial fermentation medium and the fermentation feed medium were as described in Example 1.

TABLE 5 Reb DM Phase I Length of Phase II Yield Reb DM rate phase I rate Reb D Reb M Reb DM g/g Rate D/M Biomass n= Treatment (u in h⁻¹) (hrs) (g Dx/L/hr) g/L g/L g/L Dx*100 g/L/h Ratio g DCW/L 1 Control 0.12 23 7.71 1.38 2.41 3.79 1.23 0.031 0.57 101.8 (phase II feed rate A) 2 Phase II 0.12 12 2.83 1.26 1.79 3.05 1.69 0.025 0.71 97.8 feed rate B 2 Phase II 0.12 16 4.51 1.45 1.97 3.42 1.76 0.028 0.74 101.6 feed rate C 1 Batch (all n/a n/a n/a 0.07 0.06 0.13 0.04 0.001 1.13 9.8 glucose included at start)

The phase I feed rate is kept constant and the feed phase II rate are variable but lower than used in Example 1. The data above shows improved yields with lower phase II feeding rates.

Example 3

For inoculum preparation, the yeast strain C was cultured as described in Example 1 using the seed flask medium of Table 1, Example 1. The initial fermentation medium unlike Example 1 did not include cobalt, molybdate and borate, only vitamin and trace minerals were added in the initial fermentation medium and not in the fermentation feed medium.

TABLE 6 Reb D + Treatment Phase I Phase II Reb M Reb D + Fermenta- (phase I/ rate Length of feed rate Reb Reb Reb D + Yield Reb M Reb D/ tion Biomass phase II μ in phase I g D M Reb M g/g Rate Reb M time g feeding) Replicates h⁻¹ hrs Dx L⁻¹ h⁻¹ g/L g/L g/L Dx*100 g L⁻¹ h⁻¹ ratio hours DCW/L u = 0.12/7.71 g n = 3 0.12 23 7.71 1.39 4.55 5.95 1.98 0.050 0.30 119.2 91.6 Dx/L/h u = 0.11/6.77 g n = 3 0.11 25 6.77-7.52 1.43 4.67 6.10 2.02 0.049 0.31 124.0 93.5 Dx/L/h adjusted up to 7.52 g Dx/L/h stepwise u = 0.11/6.77 g n = 2 0.11 25 6.77 1.82 4.80 6.62 2.16 0.047 0.38 141.2 92.0 Dx/L/h to end of fermentation

Improved yield with lower feeding rates, were observed as were higher rate with faster feeding (control).

Example 4 Production of Reb D and Reb M in Fed Batch Fermentation with Feedback Control of Glucose Feeding Based on Real Time Respiratory Quotient

For inoculum preparation, the yeast strain B was cultured as described in Example 1 using the seed flask medium of Table 1, Example 1.

For the fermentation, 75 mL of seed culture was transferred into initial fermentation medium as described in Table 2 of Example 1 with a starting volume of 0.75 liters. Temperature was maintained at 30° C. throughout. The air flow rate was 1.75 SLPM and the agitation rate was automatically controlled to increase in a stepwise manner from 400 to 900 rpm during the fermentation. The pH was controlled at pH 5.0 with 12% NH₄OH.

The medium was based on Verduyn et al (Verduyn C, Postma E, Scheffers W A, Van Dijken J P. Yeast. 1992 July; 8(7):501-17) with modifications as described in Tables 2 and 3 of Example 1. For the urea treatments, ammonium sulfate was increased to 15 g/L in the initial fermentation medium and urea was added to 39 g/L in the fermentation feed medium.

Since Saccharomyces cerevisiae is a Crabtree positive organism, it makes ethanol in the presence of very low levels of glucose, thus the concentration of glucose was kept limiting by controlling flow rates of fermentation feed medium, (as described in Table 3 of Example 1) to allow growth while minimizing ethanol production.

For the two phase feeding regime, an initial exponential phase (feed phase I) began at 10 hours with a growth rate of μ=0.12 l/h while the second phase of feeding (or feed phase II) started at 33 hours with a constant flow rate of 0.180 mls/minute. Feeding was continued until a final volume of 1.95 liters was obtained by 120 hours.

The treatment with respiratory quotient (RQ) based feedback control of feeding involved typical exponential feeding for the feed phase I. Then, at 39 hours, in the feed phase II of feeding, feedback control of glucose medium addition was measured on and feeding was then controlled by real time measurements of RQ by off-gas mass spectrometry analysis of oxygen and carbon dioxide concentrations in off-gas of fermentor vs. reference gas (air) with a Thermo Scientific Prima Pro Process MS instrument. The algorithm controlling feeding was designed to keep RQ between 1.05 and 1.25. RQ was calculated (by the mass spectrophotometer software by Thermo Scientific™GasWorks) by dividing carbon dioxide evolution rate (CER) by oxygen uptake rate (OUR) using the calculation:

OUR (mmol/L/h)=(F(L/min)×(% O_(2in)—% O_(2out))×60 min/h×1000 mmol/mol)/(100×24.45 L/mol×fermentor volume (L))

CER Calculation:

CER (mmol/L/h)=(F(L/min)×(% CO_(2in)−% CO_(2out))×60 min/h×1000 mmol/mol)/(100×24.45 L/mol×fermentor volume (L))

RQ=OUR/CER (a unitless ratio)

Reb D and Reb M yields on glucose were calculated based on total glucose utilized. Yield of Reb D and Reb M on biomass was based on cell dry weight. Biomass determination of cell dry weights was based on the filtration/oven method, which is commonly known in the art.

TABLE 7 Results Summary for Respiratory Quotient Based Feed Back Control Yield of Yield of Overall Off-gas Ethanol product on product on Specific (% of control Reb D (% Reb M (% glucose(% biomass(% Productivity (% during 60 h to Treatment of control) of control) of control) of control) of control) 132 h) Normal 2 100.0 100.0 100.0 100.0 100.0 100.0 phase feeding RQ control of 80.1 123.7 107.0 130.7 113.5 34.0 feeding “Normal 2-phase feeding” is 1^(st) phase: μ = 0.12 h⁻¹; 2^(nd) phase = 7.71 g L⁻¹ h⁻¹.

Example 5

For inoculum preparation, the yeast strain C was cultured as described in Example 1 using the seed flask medium of Table 1, Example 1. The initial fermentation medium unlike Example 1 did not include cobalt, molybdate and borate, only vitamin and trace minerals were added in the initial fermentation medium and not in the fermentation feed medium.

TABLE 8 Reb D + Phase I Phase 2 Reb M Reb D + rate Length of rate Reb D + Yield Reb M Reb D/ Fermentation u in phase I g Reb D Reb M Reb M g/g Rate Reb M time Biomass Treatment 1/h hrs DX/L/h g/L g/L g/L Dx*100 g L⁻¹ h⁻¹ ratio hours g DCW/L 2 phase scheduled 0.12 23 7.71 1.36 4.48 5.84 1.95 0.046 0.30 127.3 98.2 feeding (control) RQ target = 1.1 0.12 23 RQ = 1.1 0.91 4.23 5.14 1.67 0.055 0.21 94.8 104.8 RQ target = 1.3 0.12 23 RQ = 1.3 0.89 3.95 4.84 1.58 0.071 0.22 67.8 123.4 RQ target = 1.5 0.12 23 RQ = 1.5 0.97 4.12 5.09 1.65 0.077 0.23 65.7 118.4

The respiratory quotient (RQ, a unitless ratio) equals carbon dioxide evolution rate (CER, mmol/L/h) divided by oxygen uptake rate (OUR, in mmol/L/h). Respiratory quotient (RQ) targets were achieved by an algorithm in BioCommand software that increased or decreased glucose feed rate based on the RQ value from real-time off gas monitoring of carbon dioxide and oxygen in the exit gas of fermentation. RQ feedback control was only used in phase II of the two phase feeding. 

1. A method for producing one or more steviol glycoside(s), the method comprising steps of: (a) growing engineered yeast capable of producing the one or more steviol glycoside(s) in a medium, wherein the engineered yeast is grown at one or more growth rate(s) within a first range; and wherein a composition comprising glucose is added to the medium according to a first mode; and (b) fermenting the medium with the engineered yeast to produce the one or more steviol glycoside(s), wherein during fermenting, a composition comprising glucose is added to the medium according to a second mode that is different than the first mode, and during fermenting the yeast grown at one or more growth rate(s) within a second range, wherein the second range is less than the first range; wherein the one or more steviol glycoside(s) comprise rebaudioside M, rebaudioside D, or both rebaudioside M and rebaudioside D; and wherein the engineered yeast is a species of Saccharomyces.
 2. The method of claim 1, wherein the growth rate in step (a) is 0.06 h⁻¹ or greater.
 3. The method of claim 1, wherein in step (a) the first range is 0.06 h⁻¹ to 0.17 h⁻¹.
 4. The method of claim 1, wherein the growth rate in step (b) is 0.09 h⁻¹ or less.
 5. The method of claim 1, wherein in step (b) the second range is 0.015 h⁻¹ to 0.09 h⁻¹.
 6. The method of claim 1, wherein the growth rate in step (b) is in the range of 50-90% of a maximum growth rate in step (a).
 7. The method of claim 1, wherein in step (a) the composition comprising glucose is added to the medium according to the first mode which is a non-constant rate of feeding.
 8. The method of claim 1, wherein in step (b) the composition comprising glucose is added to the medium according to the second mode which is a constant rate of feeding.
 9. The method of claim 8, wherein the constant rate of feeding is not greater than 10 g glucose/L media/h.
 10. The method of claim 8, wherein the constant rate of feeding is in the range of 2 g glucose/L media/h to 10 g glucose/L media/h.
 11. The method of claim 1, where in step (b) a base is added to provide the medium with a pH that is higher than the pH of the medium in step (a).
 12. The method of claim 1, wherein step (a) is started at a time when there is less than 3 g/L of glucose in the medium.
 13. The method of claim 12, wherein step (a) is performed up to a time of 40 hours from the start of step (a) time.
 14. The method of claim 12, wherein step (b) is performed at a time of 30 hours or later from the start of step (a).
 15. The method of claim 1, wherein in step (a) the engineered yeast are grown to a biomass amount of at least 5 g dcw/L.
 16. The method of claim 15, wherein in step (a) the engineered yeast are grown to a biomass amount in the range of 20 g dcw/L to 60 g dcw/L.
 17. The method of claim 1, wherein in step (b) the engineered yeast do not grow to a biomass amount of greater than 180 g dcw/L.
 18. The method of claim 1, wherein the engineered yeast is Saccharomyces cerevisiae.
 19. The method of claim 1, wherein during step (a) the concentration of glucose is not greater than 5 g/L in the medium.
 20. The method of claim 1, further comprising a step of: (a) measuring respiratory quotient (RQ) of the engineered yeast at step (b) of claim 1 to determine if the RQ is within a range of from about 0.5 to about 2.0; (b) adjusting the glucose addition rate of the second mode when the RQ is outside of the RQ range; and (c) repeating steps (a) and (b) throughout the step of fermenting the medium with the engineered yeast cells. 